The present invention relates to cyclophosphamide analogs particularly useful for the suppression of tumor cells.
Since the demonstration in 1942 that nitrogen mustard was effective at inducing remission in patients with lymphoma (A. Gilman (1063) Amer. J. Surg. 105:574), several thousand structural analogs have been synthesized in an attempt to enhance the selectivity of the parent drug. However, only a few of these compounds have demonstrated sufficient therapeutic superiority to nitrogen mustard in experimental tumor systems to warrant clinical trail. Of these, cyclophoshamide is unquestionably the most important. It has a higher therapeutic index than must other mustard-type alkylating agents and a much broader spectrum of clinical activity. However, the drug is not independently cytotoxic; it requires enzymatic activation in order to exert biologic activity. Although the biotransformation of cyclophosphamide, in vivo, is complex, the following general principles (Scheme 1) are widely accepted (D. L. Hill (1975) `A Review of Cyclophoshamide` (Charles C. Thomas, Springfield, Ill. and O. M. Friedman, et al. (1979) Adv. Cancer Chemother. 1:143).
As shown in FIG. 1
cyclophosphamide, (1-A), is oxidatively biotransformed, mainly in the liver, by cytochrome P-450 dependent mixed-function oxidases to give 4-hydroxycyclophosphamide, (2-A). This metabolite exists in equilibrium with aldophosphamide (3-A), its open-chain tautomer. Aldophosphamide is labile and undergoes as E2 elimination reaction to generate phosphorodiamidic mustard (5-A) and acrolein (6-A). 4Hydroxycyclophosphamide and aldophosphamide also undergo further enzymatic oxidation, the former mediated by alcohol dehydrogenases and the latter by aldehyde dehydrogenases or aldehyde oxidases, to give, respectively, 4-ketocyclophosphamide (4-A) and carboxyphosphamide (7-A). Compounds 4-A and 7-A are chemically stable and relatively non-toxic. Phosphorodiamidic mustard (5), a potent alkylating agent, is generally considered to be the ultimate `active metabolite` of cyclophosphamide.
Although widespread agreement exists on the metabolism of cyclophosphamide, its mechanism of antitumor selectivity has been controversial. However, strong evidence has recently been presented in favor of the Selective Detoxification Hypothesis. The key feature of this hypothesis, first proposed by Sladek ((1973) Cancer Res. 33:1150), and later by Connors, et al. ((1974) Biochemical Pharmacol. 23:114), and Cox, et al. ((1975) Cancer Res. 35:3755), is that the conversion of aldophosphamide to carboxyphosphamide, a biologically inert compound, is less efficient in tumor cells than in most drug-susceptible normal cells (e.g., hematopoietic stem cells) because the latter contain higher levels of aldehyde dehydrogenases. As a consequence, more aldophosphamide dissociates to the highly cytotoxic phosphorodiamidic mustard in tumor cells. It has now been demonstrated that intracellular levels of aldehyde dehydrogenases are, indeed, an important biologically-operative determinant of the antitumor selectivity of cyclophosphamide. Thus, Hilton and Colvin have shown (J. Hilton, et al. (1984) Proc. Amer. Assoc. Cancer Res. 25:339) that intracellular levels of aldehyde dehydrogenase correlate inversely with cyclophosphamide sensitivity both in a variety of human and rodent hematopoietic cell lines, and in human leukemic cells; high aldehyde dehydrogenase levels were present in drug-resistant cells. An L1210 resistant cell-line with unusually high aldehyde dehydrogenase activity was rendered drug-sensitive (J. Hilton (1984) Biochem. Pharmacol. 33:1867) by pretreating the cells with low concentrations of disulfiram, an aldehyde dehydrogenase inhibitor. Equally significant, 4-hydroxycyclophosphamide was extensively converted to carboxyphosphamide, an inactive metabolite, when incubated with extracts from the drug resistant L1210 cell-line (J. Hilton (1984) Cancer Res. 44:5156). By contrast, negligible levels of carboxyphosphamide, were formed when 4-hydroxycyclophosphamide was incubated, under the same conditions, with extracts from the drug-senitive cell line. The author concluded (J. Hilton (1984) Cancer Res. 4:5156): `4-Hydroxycyclophosphamide and/or aldophosphamide is the form in which cyclophosphamide reaches these tumor cells in mice and that intracellular aldehyde dehydrogenase activity is an important determinant of cyclophosphamide sensitivity in these cell lines`.
Sladek has reported (N. E. Sladek, et al. (1985) Cancer Res. 45:1549) that three known (and one suspected) inhibitors of aldehyde dehydrogenase activity [disulfiram, diethyl dithiocarbamate, cyanamide, and (ethylphenyl (2-formylethyl) phosphinate)] potentiate the cytotoxicity of 4-hydroperoxycyclophosphamide and ASTA Z 7557 (Conference proceedings published in: (1984) Investigational New Drugs 2:1-259), (both latent precursors of 4-hydroxycyclophosphamide) when incubated against cyclophosphamide-resistant L1210 and P-388 cell-lines. Significantly, no potentiation was observed with phosphordiamidic mustard, the presumed active metabolite of cyclophosphamide. In further studies, Sladek has shown (F. R. Kohn, et al. (1984) Proc. Amer. Assoc. Cancer Res. 25:289); (F. R. Kohn, et al. (In press) Biochem. Pharmacol) that aldehyde dehyrogenase activity is an important determinant of the differential sensitivities of murine pluripotent hematopoietic stem cells and granulocyte-macrophage myeloid pregenitor cells to various activated cyclophosphamide analogs, including 4-hydroperoxycyclophosphamide and ASTA Z 7557. This finding likely accounts for the relative sparing effect of cyclophosphamide on myeloid stem cells.
Friedman, et al. (O. M. Friedman (1979) Adv. Cancer Chemother. 1:143) and, more recently, Zon (G. Zon (1982) Progress in Medicinal Chemistry 19:205) have strongly emphasized the need for further investigations in the mechanism of selectivity of cyclophosphamide and its analogs. The present application relates to new information that is critically relevant to this question. An important advantage of the present invention is the incorporation of structural and mechanistic features that contribute to the selectivity of cyclophosphamide into other antitumor drugs to enhance their therapeutic efficacy.
Advances in the treatment of acute myeloid leukemia in adults has generally been due to the introduction of new cytostatic drugs. The most important of these have been arabinosyl cytosine (Ana-C), the anthracyclines, and m-AMSA. Different combinations of these drugs give remission rates of about 60-70% (R. P. Gale (1977) Lancet 1:497); (J. F. Holland, et al. (1976) Arch Intern Med. 136:1377); and (K. B. McCredie, et al. (1981) Proc ASCO and AACR 22:479); however, the median duration of complete remission is less than 18 months, with a "cured" fraction of less than 20%.
In contrast, long-term release-free survival can be achieved in about 50% of AML-patients after high-dose chemotherapy and total body irradiation followed by allogeneic bone marrow transplantation in first remission (R. A. Clift, et al. (1985) Blood 66(5):887 (Abstract); (A. Fefer, et al. (1983) Blood 57:421); and, (K. G. Blume, et al. (1980) N. Engl. J. Med. 302:1041). Similar results have been obtained in patients with relapsing or refractory acute leukemia who receive bone marrow transplantation from an identical twin, after supralethal chemoradiotherapy (R. L. Powles, et al. (1980) Lancet 1:1047). Unfortunately, only about 25% of all patients have an HLA-compatible sibling available or bone marrow donation. The patient's own bone marrow can, however, be harvested in complete remission, cryopreserved, and used as a source of syngeneic hematopoietic stem cells for graftment purpose. This procedure allows a transplantation conditioning regiment with high-dose chemo-or chemoradiotherapy aimed at eradicating dormant leukemic cells in sanctuary sites like testicles, ovaries and the central nervous system. The problem that prevents more widespread use of cyropreserved autologous bone marrow is the presence of occult clonogeneic leukemic cells in remission bone marrow. Thus, results obtained with autologous bone marrow transplantation for AML in first remission do not differ significantly from that obtained with chemotherapy alone (A. Fefer, et al. (1983) Blood 57:421). At M. D. Anderson 10 evaluable patients were treated in second remission, with high-dose chemotherapy followed by autologous marrow transplant. Of those, one is alive in remission at 30 months, seven have relapsed (range 1-8 months) and two died early. The feasibility of using in vitro immunologic or pharmacologic treatment of remission bone marrow to eliminate occult leukemic clonogeneic cells capable of causing relapse of the disease has been convincingly proven in animal model systems (P. Stewart, et al. (1985) Exp. Hematol. 13:267); (S. J. Sharkis, et al. (1980) Blood 55:521); (H. Coizer, et al. (1982) Proc AACR 23:194); (M. Korbling, et al. (1982) Br. J. Haematol 52:89); and, (S. Thierfelder, et al. (1977) Eur. J. Cancer 15:1357). Early data for in vitro treatment ("purging") of human remission bone marrow indicate that methodology can be designed that allows successful engraftment of the patients with in vitro manipulated marrow. The available methods that have been used so far include:
(a) treatment of bone marrow with antibodies plus complement; PA0 (b) treatment with antibodies linked to immunotoxin e.g. ricin; PA0 (c) pharmacologic treatment with an in vitro active drug. PA0 (1) to develop a model for in vitro treatment of human bone marrow, obtained from patients with acute myeloid leukemia in complete remission, with a novel series of in vitro active oxazaphosphorines, PA0 (2) to determine the optimal condition under which maximum leukemic clonogeneic cell kill can be achieved with sparing of hemopoietic regenerative capacity, PA0 (3) to examine possible quantitative differences between myeloid leukemic and normal hemopoietic stem cells in the make-up of activating and degrading enzymatic machinery responsible for the resulting cytotoxicity, and PA0 (4) to explore different avenues of manipulating cellular aldehydrogenase activity, thereby augmenting differences in cytotoxicity between normal and leukemic clonogeneic stem cells.
The major weakness with the immunological "purging" methods is the lack of proven specific acute leukemia antigens that would distinguish leukemic cells from normal hemopoietic stem cells. Another technical problem is the limited availability of large quantities of monoclonal antibodies for in vitro treatment of large volumes of bone marrow.
For pharmacologic purging, the ideal drug(s) should preferably selectively kill leukemic stem cells while leaving the normal stem cells intact to allow for hemopoietic reconstitution. Obviously, such techniques alleviate the problem of finding specific anti-leukemia antibodies. Another advantage is that drug can easily be manufactured in large quantities under standardized conditions. One drug that has a possible selective action against leukemic versus normal cells is cyclophosphamide. Its in vitro active congener 4-hydroperoxycyclophosphamide, has recently received much attention for purging purposes both in murine models (P. Stewart, et al. (1985) Exp. Hematol. 13:267); (S. J. Sharkis, et al. (1980) Blood 55:521); (H. Coizer, et al. (1982) Proc AACR 23:194); (M. Korbling, et al. (1982) Br. J. Haematol 52:89); (S. Thierfelder, et al. (1977) Eur J. Cancer 15:1357); (E. S. Vitetta, et al. (1982) Immunol Rev. 62:160) and in a clinical setting (A. Hagenbeck and A. C. M. Martens (1981) Exp. Hematol 10 (Suppl 11):14); (H. Kaizer, et al. (1981) Exp Haematol 9 (Suppl 372):190) and, (L. Douay, et al. (1982) Exp Hematol 10 (Suppl 12):113.
The major shortcomings of 4-hydroperoxycyclophosphamide (4-HC) is that it has a relatively short half-life in vitro (less than 2 hrs) and that its toxic action decreases with increasing cell concentration. Furthermore, the supply of doses is limited. To circumvent these short-comings, a new series of in vitro active oxazaphosphorines is a subject of the present invention. The present application relates to investigating the in vitro activity of these compounds in human myeloid leukemic cell lines that have been developed and recently characterized, both the parent lines and sublines resistant to two of the other major anti-leukemic drugs, adriamycin and m-AMSA in comparison to their action on normal committed myeloid stem cells and pluripotent hemopoietic stem cells. A long-term goal of the present invention enables the techniques that may be applied in clinical setting for autologous bone marrow transplantation.
Among the objectives of the present invention are: